[Precise identification of O-linked β-N-acetylglucosamine peptides based on O-mesitylenesulfonylhydroxylamine...

O-linked β-N-acetylglucosamine (O-GlcNAc), an important post-translational modification of proteins, plays an essential role in maintaining normal biological activities of organisms. Studies have shown that the disruption of O-GlcNAc homeostasis is closely associated with various human diseases. Therefore, large-scale enrichment and identification of O-GlcNAc proteins is important for exploring diagnostic biomarkers of clinical diseases. O-GlcNAcylation is substoichiometric, and its glycosidic bond is unstable; hence, the enrichment and identification of O-GlcNAc proteins remains a challenge. Recently, metabolic labeling technology with per-O-acetylated unnatural monosaccharides has been introduced to enrich O-GlcNAc proteins or peptides in cells. However, azidosugars can also react with the thiol group of cysteine to form cysteine thiol-azidosugar artificial modification as a byproduct in cell metabolism, which interferes with the identification of O-GlcNAc proteins or peptides. Therefore, the development of a methodology for the specific and complete removal of cysteine thiol-azidosugar artificial modification is necessary. O-Mesitylenesulfonylhydroxylamine (MSH) is an oxidizing and aminating reagent with great potential to oxidatively eliminate the cysteine thiol modification to dehydroalanine. Hence, it is worthwhile to probe whether MSH can eliminate cysteine thiol-azidosugar artificial modification. Two cysteine thiol-azidosugar artificial modifications were synthesized successfully by incubating thiol standard peptides with tetraacetylated N-azidoacetylgalactosamine (Ac₄GalNAz) in sodium carbonate buffer (200 mmol/L, pH 10) at 37 ℃ for 90 min. Then, the reaction conditions for MSH oxidative elimination were optimized to completely remove cysteine thiol-azidosugar artificial modification. Sodium phosphate buffer (50 mmol/L, pH 8.0) was selected to protect the O-GlcNAc modification due to its mild nature. After extensive investigation, the optimized reaction conditions were established as 95 ℃ (reaction temperature) and 30 min (reaction time). Both cysteine thiol-azidosugar artificial modifications could be removed completely under these conditions. Furthermore, two azide-labeled O-GlcNAc (N₃-O-GlcNAc) peptides were used to assess whether MSH destroyed the O-GlcNAc modification at the same time. The results showed that the two N₃-O-GlcNAc peptides were stable after treatment with MSH at 95 ℃ for 30 min. In short, with excess MSH, the N₃-O-GlcNAc peptides were stable, but the cysteine thiol-azidosugar artificial modification was exhausted in sodium phosphate buffer (50 mmol/L, pH 8.0) at 95 ℃ for 30 min. Moreover, both O-GlcNAc modification and cysteine thiol-azidosugar artificial modification exist in cell metabolism. This method can not only remove cysteine thiol-azidosugar artificial modification but also ensure the stability of O-GlcNAc modification. There is growing evidence that O-GlcNAcylation mainly occurs in proteins in the cytoplasm and nucleus, and that most O-GlcNAc proteins are involved in important biological signaling pathways. The nuclear and cytoplasmic proteins incubated with Ac₄GalNAz in HeLa cells were selected as a model system. After the nuclear and cytoplasmic proteins were digested into peptides, MSH was applied to remove cysteine thiol-azidosugar modification. In addition, 51 peptides for the elimination of the cysteine thiol-azidosugar artificial modification were identified, indicating that MSH can remove the interference of cysteine thiol-azidosugar artificial modification in cell metabolism. Biotin probe and streptavidin dynabeads were subsequently used to label and enrich N₃-O-GlcNAz peptides in cell metabolism. Finally, 157 O-GlcNAc peptides attributed to 130 proteins were identified. To better understand the functional roles of O-GlcNAc proteins, gene ontology analysis was performed. Cell component analysis showed that the identified O-GlcNAc proteins were mainly distributed inpostsynaptic density, cytoplasm, and condensed nuclear chromosome. The proteins were mostly involved in biological processes, including cell division, excitatory postsynaptic potential, and microtubule-based movement. The proteins responsible for transferase activity, transferring acyl groups, histone acetyltransferase activity, and microtubule binding were highly enriched, indicating that O-GlcNAc proteins play important roles in cells. In summary, this work developed an approach to enrich O-GlcNAc peptides precisely in metabolic labeling by removing cysteine thiol-azidosugar artificial modification with MSH. This methodology provides a new strategy for the application of metabolic labeling technology with unnatural monosaccharides in glycoproteomics analysis.